1. Field of the Invention
The present invention relates to a method for diagnosing cancer by quantitative analysis of a glycoprotein which is aberrantly glycosylated in accordance with the occurrence of cancer.
2. Description of the Related Art
Proteins are involved in various life-supporting activities and post-translationally modified by signal transmission whenever necessary. The most representative post-translational modification processes are glycosylation and phosphorylation. In particular, regarding glycosylation of a glycoprotein, many monosaccharides existing on the surface of cell membrane are passed through the cell membrane by signal transduction, leading to glycosylation of a required protein by N-acetylglucosaminyltransferase. Such glycoprotein plays an important role as being located on the outer membrane. Once glycoproteins finish their required role, they proceed to glucolysis by glycosidase. However, many glycoproteins or glycolipids located on the surface of cell membrane often experience aberrant glycosylation by the specific signal such as oncogene, etc. Many diseases have been known to be closely related to such abnormal functions of glycosidase and glycosyltransferase triggered by abnormal signal transduction by oncogene (Kim, Y. J., et al., Glycoconj. J., 1997, 14, 569-576., Hakomori, S., Adv. Cancer Res., 1989, 52, 257-331., Hakomori, S., Cancer Res., 1996, 56, 5309-5318).
In the aberrant glycosylation pattern observed in cancer cells, changes in the size of N-linked glycosylation, the number of side chains, and the increase of sialylation and fucosylation are observed along with the changes in the size of glycan chain by polylactosamine formation. Such glycoprotein, thus, can be used as a cancer marker for the identification of cancer and confirmation of cancer progression by taking advantage of the said pattern observed in the aberrant glycosylation. Such aberrant glycosylation affects functions of the glycosylated protein such as folding, recognition, and solubility, etc by the specificity of the structure and the size of glycan chain (Varki, A. et al., Glycobiology 1993, 3, 97-130., Parodi, A. J. et al., Annu. Rev. Biochem. 2000, 69, 69-93). In particular, such glycotransferase as N-acetylglucosaminyltransferase that is abnormally activated in cancer cells induces the aberrant glycosylation which facilitates the distinguishment of cancer cells from normal cells, suggesting that the aberrantly glycosylated glycoprotein can be used as a marker for cancer diagnosis (Dennis, J. W.; Laferte, S.; Waghorne, C.; Breitman, M. L.; Kerbel, R. S. Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 1987, 236: 582-585). The glycoprotein harboring information on cancer is secreted to extracellular media once it completes its role therein, or is fallen off the cell membrane and shed to media. Therefore, diverse culturing media for cancer cells, cancer tissue lysis, and patient's blood samples can be proper materials for detection of the glycoprotein containing information on cancer, which can be used as a marker for cancer diagnosis.
Difference of glycosylation in protein samples obtained from the normal group and the cancer patient group can be an important clue to distinguish patient group from normal group. Up to date, many analysis methods have been reported to tell the difference of a glycoprotein. Glycan profiling is an example of the methods to screen difference in glycosylation of a protein, which analyzes glycans obtained from hydrolysis of glycoproteins by using mass spectrometer (Barkauskas. et al., Bioinformatics, 2009, 25, 251-257). However, this method has a disadvantage of losing information on the glycosylated isoform in relation to characteristics of glycosylation and glycosylated structure in the specific glycosylation site of each protein because this method only enables mass analysis of numbers of glycosylated isoform mixtures composed of all different isoforms originated from different proteins and glycosylation sites but having equal weights.
Glyco-capturing method using hydrazide has been used for the enrichment of a glycoprotein (Zhang H. et al., Nat. Biotechnol., 2003, 21, 660˜666). However, this method has a disadvantage of losing information on specific structured aberrant glycosylation derived from cancer cells because normal glycosylation structure cannot be maintained during glycan-capturing.
To separate and enrich a glycoprotein or a glycopeptide alone, considering different structures of glycan chain, ConA (Concanavalin A), WGA (Wheat germ agglutinin), Jacalin, SNA (Sambucus nigra agglutinin), AAL (Aleuria aurantia lectin), L-PHA (Phytohemagglutinin-L), PNA (Peanut agglutinin), LCA (Lens culimaris agglutinin-A), ABA (Agaricus biflorus agglutinin), DBA (Dolichos biflorus agglutinin), DSA (Datura stramonium agglutinin), ECA (Erythrina cristagalli agglutinin), SBA (Soybean agglutinin), SSA (Sambucus sieboldiana agglutinin), UEA (Ulex europaeus agglutinin), VVL (Vicia villosa lectin), BPL (Bauhinia purpurea lectin), or multilectin prepared by combinations of the above lectins can be used (Yang, Z. et al., J. Chromatogr, A, 2004, 1053, 79-88., Wang, Y. et al., Glycobiology, 2006, 16, 514-523). This is the method to utilize the selectivity of lectin to glycan chain structure of a glycoprotein, so that selective separation and enrichment of a glycoprotein having specific glycan chain structure can be possible. In particular, complexity of the target sample can be significantly reduced by eliminating such proteins that do not show affinity to lectin through the separation process of glycoproteins selective to lectin. The separated and enriched glycoproteins can be analyzed qualitatively and quantitatively by using various analysis methods.
One of those methods for analyzing a glycoprotein by using the selectivity of lectin to glycan chain structure of a glycoprotein is lectin-blotting. This method is generally performed along with immunoblotting characterized by high selectivity against certain proteins. At this time, an antibody against the antigen protein is required. If the corresponding antibody is not prepared, the protein cannot be analyzed by this method. Lectin-blotting utilizes gel-separation technique, suggesting that there is still a disadvantage of limited analyzing speed and questions remain on liability in quantitative analysis. When array technique using an antibody and lectin is used, analyzing speed and sensitivity can be significantly improved, compared with the conventional lectin-blotting (Forrester, S. et. al., Low-volume, high-throughput sandwich immunoassays for profiling plasma proteins in mice: identification of early-stage systemic inflammation in a mouse model of intestinal cancer. Mol Oncol 2007, 1(2): 216-225). However, this technique requires a liable antibody and it is very difficult to obtain each antibody against every glycoprotein newly identified at a massive scale.
In the meantime, mass spectrometry is a very useful method for high-speed, high-sensitivity qualitative and quantitative analysis of very complicated proteome samples. In particular, multiple reaction monitoring is the method to analyze quantitatively, with high liability, polypeptides having comparatively small molecular weights which are usually generated by hydrolysis of a protein. This method is especially useful when an antibody against the target protein cannot be obtained (Kuhn, E. et. al., Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics 2004, 4(4): 1175-1186). MRM is an analysis method with high sensitivity that facilitates selective analysis of the target peptide from very complicated samples by at least one of liquid chromatography and two times of precursor mass selection and fragment ion selection from those peptides generated by hydrolysis of the target protein (Anderson L, et al., Mol. Cell Proteomics. 2006, 5, 573-588).
The sample like plasma proteome is composed of at least 50,000 constituents and the density of protein components is very dynamic (1˜1012, pg/ml). So, a biomarker candidate protein existing at a very low concentration is hard to detect and analyze quantitatively by liquid chromatography-mass spectrometry (LC/MS/MS) (Anderson N. L. et al., Mol. Cell Proteomics. 2002, 1, 845-867). To minimize complexity of the sample for efficient detection of a disease biomarker in plasma, proteins taking about 90% or more of plasma proteome such as albumin, IgG, IgA, transferrin and haptoglobin are first eliminated. And then the resultant proteome is used for analysis. Even after the minimization of the complexity of the sample by eliminating those proteins exiting at high concentrations in plasma and the increased high selectivity against the target peptide by LC-MRM, if a target marker protein exists at a very low concentration in the sample, the concentration of the marker protein or the hydrolyzed marker peptide is increased through enrichment process of the marker protein or the marker peptide by using immunoaffinity to improve LOD (limit of detection) and LOQ (limit of qualification).
The present inventors tried to develop a method for diagnosing cancer by using quantitative information on a glycoprotein which is aberrantly glycosylated in accordance with the occurrence of cancer, during which the inventors obtained polypeptides by hydrolyzing the said glycoprotein after separating and concentrating the glycoprotein, selected hydrolyzed marker peptides derived from the marker glycoprotein characteristically demonstrating cancer specific glycosylation, and finally completed this invention by confirming that the said marker peptides could be effectively used for cancer diagnosis.